Enzyme Linked Immunosorbent Assay (ELISA)
ELISA stands for enzyme linked immunosorbent assay.
ELISA (enzyme linked immunosorbent assay) is a plate based assay technique that allows for detection and quantification of an analyte present in a sample. In essence, ELISA relies on the binding of antibodies to a target antigen, thus enabling detection and quantification of the binding antigen. Even if present in small concentrations, ELISA is able to detect multiple substances, including hormones, peptides, proteins and antibodies. Therefore, it is a valuable tool for diagnosis, industry and research. While several types of ELISA have been established, they all rely on the same principles:
- Plate Coating. In direct and indirect ELISA, antigens are immobilised directly to the surface of a polystyrene microplate well. Nevertheless, primary capture antibodies can also be coated to the surface of the plate for other ELISA variants. The different types of ELISA will be discussed in detail in the following sections.
- Plate Blocking. In this step, all the surface-binding sites present in the coated plates are saturated by adding a protein or molecule that does not interfere with the assay that is being carried out. Blocking the surface area is essential to obtain reliable results as the binding capacity of the plate is usually higher than the amount of antigen or antibody coated. One of the most common blocking agents is Bovine Albumin Serum (BSA).
- Antibody Incubation. After coating, antigen-specific antibodies with high affinity to the antigens are incubated for detection. The analyte of choice is detected using a tag. While this tag can be conjugated directly to the antigen-specific antibody, it can also be included in a secondary detection antibody. In general, using more than one antibody for detection results in signal amplification as several labelled antibodies will bind to the different epitopes of the primary antibody. Common tags include enzymes such as HRP (horse radish peroxidase) and AP (alkaline phosphate). HRP, in particular, reacts with TMB (tetramethylbenzidine) and produces a change in colour that can be used to determine the concentration of the analyte. Nevertheless, antibodies can also be conjugated to fluorescent tags.
- Detection and signal measurements. Signal detection and measurement varies according to the tag used. For colorimetric ELISA, HRP or AP, and their corresponding substrate, is normally used. The signal is then detected using a standard absorbance plate reader, which enables direct visualisation and high reproducibility between plates. If the ELISA has been accurately developed, the signal produced by the substrate will be proportional to the concentration of the antigen captured. For fluorescent tags, however, a fluorometer that produces the appropriate excitation beam is required. This method also allows high reproducibility. Yet, it normally required the use of black microtiter plate.
The four major types of ELISA are indirect, direct, sandwich and competitive.
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Direct ELISA is the simplest type of ELISA as it only uses one antibody for detection of the analyte of interest. Once the antigen is coated and the plate is blocked, the antigen-specific antibody is incubated. For detection, the antibody used is conjugated to an enzyme or molecule that produces a colour change upon substrate addition, which in turn, enables quantification. Direct ELISA shows minimal cross-reactivity as only one antibody is used. Yet, conjugation of the primary antibody to the enzyme increases the cost and time associated with running multiple experiments. With each new assay, a new detection antibody needs to be labelled. Compared to other ELISA variants, direct ELISA is faster, as fewer steps are required for detection. However, it exhibits lower sensitivity as there is no signal amplification.
Indirect ELISA requires the use of two antibodies for detection. This adds an extra step and increases the time required to complete the assay. Once the antigen is coated and the plate is blocked, the primary antigen-specific antibody is incubated. A labelled secondary antibody, that binds to the primary antibody, is then added for detection and subsequent measurement. The concentration of the analyte can be determined using the absorbance measurements obtained from the secondary antibody. Compared to direct ELISA, this approach exhibits higher sensitivity as each primary antibody contains several epitopes that can be bound to the labelled secondary antibody, resulting in signal amplification. Flexibility may also be increases as more than one secondary antibody can be used with a single primary antibody.
Competitive ELISA is the most complex type of ELISA and all variants can be adapted to perform in a competitive format. Competitive ELISA s mainly used to identify small molecules such as lipids, hormones, and small peptides. This assay follows the principle that the antigen of interest and a conjugated version of the same antigen will compete for the binding sites present on the antibody. Therefore, the higher the sample antigen concentration, the weaker the signal output will be. The signal output is then inversely proportional to the amount of antigen in the sample. The labelled antigen will compete for binding to the antibody pre-coated to the wells. If more of the antigen in the sample is able to bind to, more of the labelled antigen will be removed following the wash cycles, and consequently, lower absorbance values will be obtained.
Sandwich ELISA is the most common type of ELISA. As the name suggests, the antigen of interest is sandwiched between two antibodies either directly or indirectly. This type of assay requires the use of matched pairs of antibodies that have been tested together to ensure that they bind different and non-overlapping epitopes of an antigen, thus preventing the antibodies binding to the same site. The capture antibody is coated on the surface of the plate and binds to the target antigen after the sample is added. The detection antibody, on the other hand, binds to the target antigen if it is has bound to the capture antibody and enables subsequent measurements either directly or indirectly. If the detection antibody is enzyme-conjugated, the assay is a direct sandwich ELISA. Contrarily, if the detection antibody us not labelled we are referring to an indirect sandwich ELISA and another enzyme-conjugated antibody would be required for detection.
Sandwich ELISA shows greater sensitivity and specificity as two antibodies are used to detect a single antigen. In general, it is a useful tool to analyse complex samples as they do not need to be purified prior to the assay. Similarly, sandwich ELISA is particularly useful when samples are suspected to have low concentrations of the antigen of interest. If standardised ELISA kits are not available for the analyte of interest, optimisation of the antibody pairs can be difficult due to cross-reactivity.
Refer to our ELISA protocols for more information.
Several buffers are employed in any time of ELISA: for coating, for blocking, for washing, and for sample or antibody dilutions. Each of these buffers can be produced in house or provided from a supplier. The basic recipe for each of the mentioned buffers can be found on our ELISA protocol. Alternatively, you can find our full list of ELISA reagents here.
As discussed, coating is the first step in ELISA and involves the incubation of a relevant antigen or antibody until it is immobilised to the surface of the well. Immobilisation occurs passively because of the hydrophobic interactions between amino acid side chains on the antibody or antigen, and the plastic surface. Therefore, it can be influenced by external factors such as incubation time, temperature, or pH and concentration of the coating buffer.
In general, the coating process requires the addition of 100 µl of the coating buffer with the selected antigen or antibody to a concentration of 1-20 µg/ml, and incubation overnight at 4°C overnight or for ~2 hours at room temperature. High concentrations of the antigen or antibody may negatively affect coating due to oversaturation of the wells and consequently, impaired binding. Thus, it is important to check the manufacturer's recommended concentrations for ELISA, or to test different concentrations for each assay. Insufficient coating of the antigen or antibody to the microtiter plates can result in false negative results. Therefore, it is essential to maintain a humid environment in the wells to avoid evaporation. Plate sealers are usually used to maintain consistent conditions throughout the assay.
The most common coating buffers for ELISA include Phosphate Buffered Saline (PBS) and bicarbonate buffer at pH 9.6.
Blocking buffers are used to prevent non-specific binding of the detection antibodies to the surface of the coated plated. Unwanted proteins and other biomolecules may bin to unoccupied space on the surface of the well, which can be detrimental to the specificity and sensitivity of ELISA as less antigen will be able to bind. The two major classes of blocking reagents are proteins and reagents. Protein-based blocking buffers are permanent blocking agents. Tus, they must be added after the capture antibody has been immobilised. Contrarily, detergent-based buffers only block temporarily and are washed away during the washing cycles. When choosing a blocking agent, it is important to consider the following.
- It exhibits no cross-reactivity with any other component involved in the assay. Therefore, it is important to select a buffer that contains an unrelated protein that does not react with any of the antibodies used for detection.
- It helps to stabilise biomolecules by minimising the effects of denaturation that may arise between steps.
- It exhibits low enzyme activity and prevents any interference with the detection method.
The most common protein blocking buffers contain 0.3-5% Bovine Albumin Serum (BSA) dissolved in PBS because BSA blocking buffers have high affinity for proteins. Other forms of protein blockers include non-fat dry milk, whole natural serum, and fish gelatin. Tween 20, on the other hand, is one of the most common blocking agents. It is a non-ionic detergent that can block areas of the plate surface that may be exposed. Tween 20 is cheap and very stable, and can also be employed for washing buffers. In general, ELISA plates are blocked with 150-200 µl of the blocking buffer per well and incubated at room temperature for 1-2 hours.
Washing is one of the key steps to optimise ELISA as it reduces the background signal associated with unbound materials and conjugated antibodies. Washing involves filling the wells with a washing buffer, which is usually PBS with Tween 20. Typically, wells are washed 3-5 times between each step with 200-300 µl of the washing buffer. The washing solution is usually retained in the wells for about 1 minute and aspirated to prevent the dilution of the reagents added in the subsequent stages.
ELISA Detection Methods
As discussed, ELISA rely on the signal generated by an enzymatic label, which in turn, correlates with binding of an antibody to its antigen. Several enzymes and substrates are available for ELISA as well as several methods on which these can be incorporated into the assay.
The most common type of ELISA detection method uses a colorimetric approach. HRP and AP conjugated antibodies are used in combination with a chromogenic substrate like TMB, which produces a change in colour. Once the colour change is observed, the absorbance is recorded and compared between samples. Nevertheless, the concentration of bound antigen in each sample can also be calculated using a standard curve. A second methodology uses enzymes like HRP or AP with fluorescent substrates. In this case, the fluorescent signal is measured with a fluorometer to determine the concentration of the antigen. Yet, it is important to consider that fluorescent labels have a shorter half-life. Similarly, fluorescent assays require the use of black plates to minimise the background signal and obtain accurate measurements.
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